Monocarboxylate transporter 4 (MCT4) antisense oligonucleotide (ASO) inhibitors for use as therapeutics in the treatment of cancer

ABSTRACT

Provided herein are compositions, method and uses for modulating MCT4 activity or for the treatment of cancer. The compositions comprise antisense oligonucleotides (ASO) for administration to a cancer cell, wherein the cancer cell may be characterized by elevated expression of MCT4. The cancer may be selected from one or more of: prostate cancer; renal cell carcinoma; breast cancer; cervical cancer; liver cancer; bladder cancer; and small cell lung cancer pr. The prostate cancer may be castration-resistant prostate cancer (CRPC).

CROSS REFERENCE TO RELATED APPLICATION

This application is a 35 U.S.C. 371 national stage entry ofInternational Application No. PCT/CA2016/000296, filed 30 Nov. 2016,which application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/260,837 filed on 30 Nov. 2015, entitled“ANTISENSE OLIGONUCLEOTIDES AS MONOCARBOXYLATE TRANSPORTER 4 THERAPEUTICCOMPOSITIONS AND METHODS FOR THEIR USE IN THE TREATMENT OF CANCER”,which applications are incorporated herein by reference in theirentireties and for all purposes.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith as a text file, “CARB-024 SeqList ST25.txt” created on May 23, 2018 and having a size of 8 KB. Thecontents of the text file are incorporated by reference herein in theirentirety.

TECHNICAL FIELD

The present invention provides compounds, compositions and methods formodulating the expression of monocarboxylate transporter 4 (MCT4). Inparticular, this invention relates to antisense oligonucleotides (ASOs)capable of modulating human MCT4 mRNA expression, and their uses andmethods for the treatment of various indications, including variouscancers. In particular the invention relates to therapies and methods oftreatment for cancers such as prostate cancer, includingcastration-resistant prostate cancer (CRPC).

BACKGROUND

Prostate cancer is the most common non-cutaneous cancer and the secondleading cause of cancer-related deaths for males in the Western world(Siegel R, et al., 2012., 62(1):10-29). Prostate cancers are initiallyandrogen-dependent, and while androgen deprivation therapy (ADT) caninduce marked tumor regression, resistance to ADT inevitably emerges,leading to castration-resistant prostate cancer (CRPC). The currentstandard care for treating CRPC is systemic, docetaxel-basedchemotherapy, increasing the overall survival of patients by about 2months compared to mitoxantrone-based therapy (Petrylak D P, et al., NEngl J Med. 2004; 351(15):1513-1520; Tannock I F, et al., N Engl J Med.2004; 351(15):1502-1512). Recently, sipuleucel-T, cabazitaxel,abiraterone, MDV3100 and Radium-223 have shown more prolonged overallsurvival benefit and are approved by the FDA for treatment of the CRPC(Bishr M and Saad F., Nat Rev Urol. 2013; 10(9):522-528). Although theefficacy of metastatic castration-resistant prostate cancer (mCRPC)treatment has recently been improved by using more powerfulchemotherapeutics targeting the androgen receptor (AR)-signaling axis,such as enzalutamide (Ning et al., 2013) and abiraterone (de Bono etal., 2011), the overall survival of patients has only marginallyincreased (Badrising et al., 2014; Loriot et al., 2013; Tannock et al.,2004). Moreover, it is thought that further improved versions of suchdrugs can promote transdifferentiation of prostatic adenocarcinoma toneuroendocrine prostate cancer (NEPC), a subtype of the disease that iscurrently incurable (Beltran et al., 2011; Nadal et al., 2014; Yuan etal., 2007) However, none of these drugs are curative and theyincrementally improve overall survival. The establishment of moreeffective therapeutic targets and drugs, specifically those targetingthe molecular drivers of metastatic CRPC and mCRPC, is of criticalimportance for improved disease management and patient survival (Lin D,et al., Curr Opin Urol. 2013; 23(3):214-219).

There is increasing evidence that targeting reprogrammed energymetabolism of cancers offers a unique approach for effective therapeuticintervention (Zhang and Yang, 2013). For glucose utilization, cancercells, as distinct from normal resting cells, in general have apreference for glycolysis coupled to lactic acid production, i.e. aprocess called aerobic glycolysis or the Warburg effect (Warburg, 1956).This leads to elevated glucose consumption, a near-universal property ofprimary and metastatic cancers. In addition, aberrant utilization ofglutamine, also leading to elevated lactic acid production, has beenobserved to be highly common for cancers (Koochekpour et al., 2012).These metabolic energy pathways lead to increased lactic acid secretionby the cancer cells into their microenvironment, facilitating multipleoncogenic, lactate-stimulated processes, including tissueinvasion/metastasis, neo-angiogenesis and responses to hypoxia (Choi etal., 2013; Choi et al., 2014; Doherty and Cleveland, 2013; Ullah et al.,2006). Furthermore, lactic acid-induced acidification of the cancer cellmicroenvironment (to pH 6.0-6.5) can lead to suppression of local hostanticancer immunity (Choi et al., 2013; Parks et al., 2013). Thephenomenon of enhanced glucose metabolism by cancers is most commonlyexploited clinically by 18F-fluorodeoxyglucose positron emissiontomography (FDG-PET). Although this imaging technique is not generallyused for prostate cancer (Jadvar, 2009), there is evidence suggestingthat glucose metabolism of prostate cancer cells is increased by ARsignaling and progression to treatment resistance (Tennakoon et al.,2014; Vaz et al., 2012). As such, targeting the aerobic glycolyticpathway could be effective for treating advanced prostate cancers.

The monocarboxylate transporter (MCT) family consists of plasma membranetransporter proteins involved in the transport of lactic acid and othermetabolic monocarboxylates. In particular, the cellular efflux of lacticacid/H+ is thought to be predominantly mediated by MCT4 (SLC16A3)(Dimmer et al., 2000). Expression of MCT4 has been associated withhighly glycolytic cells (Halestrap, 2013; Manning Fox et al., 2000;Ullah et al., 2006), and elevated expression of MCT4 in tumours isclinically relevant as it has been associated with poor patientprognosis in multiple types of cancer (Fisel et al., 2013; Lisanti etal., 2013; Ohno et al., 2014), including prostate cancer (Hao et al.,2010; Pertega-Gomes et al., 2011). Furthermore, elevated MCT4 expressionmay be important in cancer-stroma interactions facilitating prostatecancer progression (Sanità et al., 2014). This information, togetherwith the cancer growth-promoting ability of cancer-generated lacticacid, suggests that inhibition of the expression or function of MCTsprovides a promising therapeutic strategy for a wide variety ofneoplasms (Marchiq et al., 2015). However, a therapeutic strategyspecifically targeting MCT4-mediated efflux of lactic acid is stilllacking.

Therapeutic options for castration resistant prostate cancer (CRPC)treatment have changed considerably with the recent FDA approvals ofnewer agents that improve patient survival. In particular, Enzalutamide(ENZ), a second generation androgen receptor antagonist approved fortreating metastatic CRPC in post-docetaxel and more recently,pre-docetaxel setting. However, within 2 years of clinical practice,development of ENZ resistance was evident in majority of patients(Claessens et al. 2014) and no known therapies were shown to beeffective to ENZ-resistant CRPC to-date. Thus, a novel therapeutic agentthat can effectively suppress ENZ-resistant CRPC would be useful.

SUMMARY

The present invention is based in part on the discovery that theinhibition of MCT4 expression with MCT4 antisense oligonucleotides(ASOs) (SEQ ID NO:1-23 and SEQ ID NO:46-50) may be useful in thetreatment of cancer. The inhibition of MCT4 expression with certain ASOsleads to reduced proliferation of cancer cells. Furthermore, thatreduction in proliferation extends to castration-resistant prostatecancer (CRPC).

In a first aspect, there is provided a composition for the treatment ofa cancer cell, the method including administering one or moreoligonucleotides selected from SEQ ID NO:1-23 or SEQ ID NO:46-50 to thecell.

In a further aspect, there is provided a pharmaceutical composition, thecomposition including (a) an antisense oligonucleotide (ASO) may beselected from one or more oligonucleotides of SEQ ID NO:1-23 and SEQ IDNO:46-50; and (b) a pharmaceutically acceptable carrier.

In accordance with a further aspect, there is provided a commercialpackage including (a) an antisense oligonucleotide sequence describedherein and a pharmaceutically acceptable carrier; and (b) instructionsfor the use thereof for modulating MCT4 activity.

In a further aspect, there is provided a commercial package, including:(a) an ASO may be selected from an oligonucleotide of SEQ ID NOs:1-23 orSEQ ID NOs:46-50; and (b) instructions for the treatment of cancer.

In a further aspect, there is provided a method of treating cancer, themethod including administering an antisense oligonucleotide (ASO) whichmay have a sequence selected from one or more of: SEQ ID NOs:1-23 andSEQ ID NOs:46-49.

In a further aspect, there is provided a method of treating cancer, themethod including administering an MCT4 antisense oligonucleotide (ASO)having a sequence selected from one or more of: SEQ ID NOs:1-23 and SEQID NOs:46-49.

In a further aspect, there is provided an antisense oligonucleotide(ASO), wherein the oligonucleotide may have a sequence selected from thefollowing: (a) TCCCATGGCCAGGAGGGTTG (SEQ ID NO:1); (b)GTCCCGGAAGACGCTCAGGT (SEQ ID NO:2); (c) AAGGACGCAGCCACCATGCC (SEQ IDNO:3); (d) TTGGCGTAGCTCACCACGAA (SEQ ID NO:4); (e) AGATGCAGAAGACCACGAGG(SEQ ID NO:5); (f) CCACTCTGGAATGACACGGT (SEQ ID NO:6); (g)GTAGGAGAAGCCAGTGATGAC (SEQ ID NO:7); (h) AGCATGGCCAGCAGGATGGA (SEQ IDNO:8); (i) GGCTGGAAGTTGAGTGCCAA (SEQ ID NO:9); (j) CATGCCGTAGGAGATGCCAA(SEQ ID NO:10); (k) CTCAGGCTGTGGCTCTTTGG (SEQ ID NO:11); (l)TAGCGGTTCAGCATGATGA (SEQ ID NO:12); (m) AGCACGGCCCAGCCCCAGCC (SEQ IDNO:13); (n) GAGCTCCTTGAAGAAGACACT (SEQ ID NO:14); (o)CAGGATGGAGGAGATCCAGG (SEQ ID NO:15); (p) AGACCCCCCACAAGCATGAC (SEQ IDNO:16); (q) GAAGTTGAGTGCCAAACCCAA (SEQ ID NO:17); (r)CCCGTTGGCCATGGGGCGCC (SEQ ID NO:18); (s) GCCAGCCCGTTGGCCATGGG (SEQ IDNO:19); (t) AGGAAGACAGGGCTACCTGC (SEQ ID NO:20); (u)GCACACAGGAAGACAGGGCT (SEQ ID NO:21); (v) CAGGGCACACAGGAAGACAG (SEQ IDNO:22); (w) CAGCAGTTGAGCAGCAGGCC (SEQ ID NO:23); (x) ATGGCCAGGAGGGTTG(SEQ ID NO:46); (y) CATGGCCAGGAGGGTT (SEQ ID NO:47); (z)CCATGGCCAGGAGGGT (SEQ ID NO:48); and (aa) CCCATGGCCAGGAGGG (SEQ IDNO:49).

In a further aspect, there is provided a pharmaceutical composition, thecomposition including (a) an antisense oligonucleotide (ASO) having asequence selected from one or more of: SEQ ID NOs:1-23 and SEQ IDNOs:46-49; and (b) a pharmaceutically acceptable carrier.

In a further aspect, there is provided a use of an antisenseoligonucleotide (ASO) having a sequence selected from one or more of:SEQ ID NOs:1-23 and SEQ ID NOs:46-49 for treating cancer.

In a further aspect, there is provided a use of an antisenseoligonucleotide (ASO) having a sequence selected from one or more of:SEQ ID NOs:1-23 and SEQ ID NOs:46-49 in the manufacture of a medicamentfor treating cancer.

In a further aspect, there is provided a combination therapy, thecombination comprising (a) an antisense oligonucleotide (ASO) having asequence selected from one or more of: SEQ ID NOs:1-23 and SEQ IDNOs:46-49; and (b) Docetaxel. The combination therapy may furtherinclude a pharmaceutically acceptable carrier or excipient.

In a further aspect, there is provided a commercial package, includingan ASO having a sequence selected from one or more of: SEQ ID NOs:1-23and SEQ ID NOs:46-49; and instructions for the treatment of cancer.

The antisense oligonucleotide (ASO) may have the sequence of SEQ IDNOs:1-23 or SEQ ID NOs:46-49. The antisense oligonucleotide (ASO) mayhave the sequence of SEQ ID NO:1. The antisense oligonucleotide (ASO)may have the sequence of SEQ ID NO:2. The antisense oligonucleotide(ASO) may have the sequence of SEQ ID NO:3. The antisenseoligonucleotide (ASO) may have the sequence of SEQ ID NO:4. Theantisense oligonucleotide (ASO) may have the sequence of SEQ ID NO:5.The antisense oligonucleotide (ASO) may have the sequence of SEQ IDNO:6. The antisense oligonucleotide (ASO) may have the sequence of SEQID NO:7. The antisense oligonucleotide (ASO) may have the sequence ofSEQ ID NO:8. The antisense oligonucleotide (ASO) may have the sequenceof SEQ ID NO:9. The antisense oligonucleotide (ASO) may have thesequence of SEQ ID NO:10. The antisense oligonucleotide (ASO) may havethe sequence of SEQ ID NO:11. The antisense oligonucleotide (ASO) mayhave the sequence of SEQ ID NO:12. The antisense oligonucleotide (ASO)may have the sequence of SEQ ID NO:13. The antisense oligonucleotide(ASO) may have the sequence of SEQ ID NO:14. The antisenseoligonucleotide (ASO) may have the sequence of SEQ ID NO:15. Theantisense oligonucleotide (ASO) may have the sequence of SEQ ID NO:16.The antisense oligonucleotide (ASO) may have the sequence of SEQ IDNO:17. The antisense oligonucleotide (ASO) may have the sequence of SEQID NO:18. The antisense oligonucleotide (ASO) may have the sequence ofSEQ ID NO:19. The antisense oligonucleotide (ASO) may have the sequenceof SEQ ID NO:20. The antisense oligonucleotide (ASO) may have thesequence of SEQ ID NO:21. The antisense oligonucleotide (ASO) may havethe sequence of SEQ ID NO:22. The antisense oligonucleotide (ASO) mayhave the sequence of SEQ ID NO:23. The antisense oligonucleotide (ASO)may have the sequence of SEQ ID NO:46. The antisense oligonucleotide(ASO) may have the sequence of SEQ ID NO:47. The antisenseoligonucleotide (ASO) may have the sequence of SEQ ID NO:48. Theantisense oligonucleotide (ASO) may have the sequence of SEQ ID NO:49.

The ASO may further include a modified internucleoside linkage. Themodified internucleoside linkage may be a peptide-nucleic acid linkage,a morpholino linkage, a N3′ to P5′ phosphoramidate linkage, amethylphosphonate linkage or a phosphorothioate linkage. The modifiedinternucleoside linkage may be a peptide-nucleic acid linkage. Themodified internucleoside linkage may be a morpholino linkage. Themodified internucleoside linkage may be a N3′ to P5′ phosphoramidatelinkage. The modified internucleoside linkage may be a methylphosphonatelinkage. The modified internucleoside linkage may be a phosphorothioatelinkage. The ASO may further include a modified sugar moiety. Themodified sugar moiety may be 2′-O-alkyl oligoribonucleotide. The ASO mayhave a 2′MOE gapmer modification. The ASO may further include a modifiednucleobase. The modified nucleobase may be a 5-methyl pyrimidine or a5-propynyl pyrimidine. The cell may be a human cell. The cancer may becharacterized by elevated expression of MCT4. The cancer may be selectedfrom one or more of the following: prostate cancer; renal cellcarcinoma; breast cancer; cervical cancer; liver cancer; bladder cancer;and small cell lung cancer. The cancer may be selected from one or moreof the following: prostate cancer; renal cell carcinoma; breast cancer;cervical cancer; liver cancer; and bladder cancer. The cancer may beselected from one or more of the following: prostate cancer; renal cellcarcinoma; breast cancer; cervical cancer; and liver cancer. The cancermay be selected from one or more of the following: prostate cancer;renal cell carcinoma; breast cancer; and cervical cancer. The cancer maybe selected from one or more of the following: prostate cancer; renalcell carcinoma; and breast cancer. The cancer may be selected from oneor more of the following: prostate cancer; and renal cell carcinoma. Thecancer may be selected from one or more of the following: prostatecancer; and small cell lung cancer. The cancer may be selected from oneor more of the following: prostate cancer; renal cell carcinoma; breastcancer; cervical cancer; liver cancer; and small cell lung cancer. Thecancer may be selected from one or more of the following: prostatecancer; renal cell carcinoma; breast cancer; cervical cancer; bladdercancer; and small cell lung cancer. The cancer may be selected from oneor more of the following: prostate cancer; renal cell carcinoma; breastcancer; liver cancer; bladder cancer; and small cell lung cancer. Thecancer may be selected from one or more of the following: prostatecancer; renal cell carcinoma; cervical cancer; liver cancer; bladdercancer; and small cell lung cancer. The cancer may be prostate cancer.The prostate cancer may be castration-resistant prostate cancer (CRPC).The cancer may be selected from one or more of the following: prostatecancer; renal cell carcinoma; breast cancer; liver cancer; and bladdercancer. The prostate cancer may be enzalutamide (ENZ) resistant CRPC.The cancer may be metastatic prostate cancer. The ASO may besubstantially complementary to the mRNA of MCT4. The ASO may beadministered intravenously. The ASO may be topically administered to atissue. The ASO may be mixed with lipid particles prior toadministration. The ASO may be encapsulated in liposomes prior toadministration.

The ASO may further include a modified internucleoside linkage. Themodified internucleoside linkage may be a peptide-nucleic acid linkage,a morpholino linkage, a N3′ to P5′ phosphoramidate linkage, amethylphosphonate linkage or a phosphorothioate linkage. The ASO mayfurther include a modified sugar moiety. The modified sugar moiety maybe a 2′-O-alkyl oligoribonucleotide. The ASO may further have a 2′MOEgapmer modification. The ASO may further include a modified nucleobase.The modified nucleobase may be a 5-methyl pyrimidine or a 5-propynylpyrimidine.

The cell may be a human cell. The cancer may be characterized byelevated expression of MCT4. The cancer may be selected from one or moreof the following: prostate cancer; renal cell carcinoma; breast cancer;cervical cancer; liver cancer; bladder cancer; and small cell lungcancer. The prostate cancer may be castration-resistant prostate cancer(CRPC). The prostate cancer may be enzalutamide (ENZ) resistant CRPC ormetastatic CRPC (m CRPC).

The ASO may be substantially complementary to the mRNA of MCT4. The ASOmay be administered intravenously. The ASO may be topically administeredto a tissue. The ASO may be mixed with lipid particles prior toadministration. The ASO may be encapsulated in liposomes prior toadministration. Antisense oligonucleotides and may be used asneoadjuvant (prior), adjunctive (during), and/or adjuvant (after)therapy with surgery, radiation (brachytherapy or external beam), orother therapies (e.g. HIFU). For example, MCT4 antisenseoligonucleotides may be used in combination with docetaxel, MDV3100, orwith modulators of glucose metabolism (including doxycycline or othermitochondrial inhibitors).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows MCT4 expression in tissue microarrays (TMA) frompatient-derived prostate cancer samples available at the VancouverProstate Centre Tissue Bank and stained for MCT4 expression, wherein A)and B) show bar graphs confirming that elevated MCT4 expression isassociated with high Gleason grade; C) shows plot of wherein high MCT4expression is associated with earlier time to relapse as measured byincreases in serum PSA; and D) and E) show two bar graphs representingpatients undergoing prolonged neoadjuvant hormone therapy (NHT) andthose with CRPC also show elevated MCT4 expression.

FIG. 2 shows the efficacy screening of MCT4-targeting ASOs using PC-3cells in vitro for inhibition of MCT4 expression and cell proliferation,wherein MCT4 ASOs #1 (SEQ ID NO: 1) and #14 (SEQ ID NO: 5) exert theirin vitro effects in a dose dependent manner, and the effects persistpast 96 hours after transfection: A) shows siRNA-silencing of MCT4 inPC-3 cells showed significant inhibition of cell proliferation,indicating that inhibition of MCT4 expression could have potentialtherapeutic efficacy; B) shows screening of ten MCT4-targeting ASOsrevealed varying inhibitory effects on cell proliferation, withsequences #1 (SEQ ID NO: 1) and #14 (SEQ ID NO: 5) showing the mostprofound inhibitions; C) shows MCT4 ASOs inducing various levels of MCT4knockdown as measured by qPCR and Western blot, with ASOs #1 and #14being the most effective; D) shows a strong correlation (p<0.001) wasfound between the MCT4 mRNA expression and the resulting cell numbers,indicating that the inhibitory effects on cell proliferation arestrongly related to decreased MCT4 expression (dotted lines representthe 95% confidence interval); E) wherein transfection of 5 nM to 200 nMof MCT4 ASOs shows that the inhibition of cell proliferation andexpression of MCT4 is similarly dose dependent (IC₅₀=32 nM for ASO #14,IC₅₀=50 nM for ASO #1); and F) shows a time course experimentdemonstrates that the inhibition of cell proliferation and MCT4expression following MCT4 ASO transfections persists up to at least 96hours post-transfection.

FIG. 3 shows MCT4 ASOs #1 (SEQ ID NO: 1) and #14 (SEQ ID NO: 5) areeffective in vitro against human prostate cancer cells other than PC-3,but not against mouse prostate cancer cells: A) shows candidate MCT4ASOs are also able to inhibit C4-2 human prostate cancer cellproliferation and expression of MCT4 in a dose-dependent manner withIC₅₀ values comparable to those observed with PC-3 cells; B) showscandidate MCT4 ASOs also inhibit DU145 human prostate cancer cellproliferation in a dose-dependent manner with IC₅₀ values similar tothose obtained with the other cell lines, wherein the inhibition of cellproliferation is accompanied by a decrease in MCT4 expression; and C)shows that candidate MCT4 ASOs do not have any appreciable effects onTRAMPC2 mouse prostate cancer cells and they neither affect cellproliferation nor mouse MCT4 expression levels even at the highesttested concentration of 200 nM, showing that the tested ASOs arespecific for human MCT4 and that the inhibition of cell proliferation isa phenomenon related to MCT4 knock-down.

FIG. 4 shows that transfection of candidate MCT4 ASOs leads toinhibition of glucose metabolism of PC-3 cells: A) shows that MCT4 ASOssignificantly inhibited lactic acid secretion and that correspondingaccumulation of intracellular lactate and inhibition of glucoseconsumption is also observed (u.d.=undetectable) 48 hourspost-transfection; and B) shows a schematic of the glycolysis pathwayand the changes in metabolism caused by MCT4 knock-down andcharacterized by qPCR analysis of gene expression levels of variousgenes involved in glycolysis and lactic acid conversion, wherein asignificant decrease in expression of various genes in glycolysissuggests an overall decrease in glucose metabolism and the decreasedexpression of LDHA and PDK1 suggest a redirection of pyruvate away fromlactic acid production toward the TCA cycle for oxidativephosphorylation.

FIG. 5 shows MCT4 ASO-induced reduction of MCT4 expression in PC-3tumour cells in vivo was associated with inhibition of PC-3 tumourgrowth, characterized by an increase in apoptosis and inhibition of cellproliferation, based on athymic nude mice bearing subcutaneous PC-3tumours treated with intraperitoneal injections of MCT4 ASOs #1 (SEQ IDNO: 1), #14 (SEQ ID NO: 5), control ASO, or vehicle (PBS) at 10 mg/kgdaily for 5 days followed by 2 days off treatment for a total of 15days: A) shows a plot representing percent tumour volume by treatmentday comparing MCT4 ASOs in relation to the rate of tumour growth; B)shows bar graphs representing caspase 3 positive cells and percent Ki-67positive cells following treatment with MCT4 ASOs as attributable to anincrease in cell apoptosis and a decrease in cell proliferation; and C)shows treatment with MCT4 ASOs decreased MCT4 expression in the tumouras measured by IHC staining.

FIG. 6 shows that MCT4-targeting ASOs increase immune cell aggregationand alter tumour-associated immune cell proportions in vivo (i.e. NKcells and CD3 positive cells): A) shows a bar graph wherein theimmunomodulatory effects of treatment with MCT4 ASO are partiallyexerted through a significant increase in the extent of such immune cellaggregations; B) shows a bar graph wherein the treatment with MCT4 ASOincreased the proportion of NK cells associated with the tumour; and C)shows a bar graph representing the proportion of CD3 positive cells as amarker for activated NK cells (since nude mice lack T cells) in thepresence of MCT4 ASOs as compared to control.

FIG. 7 shows that candidate MCT4-targeting ASOs are able to inhibit cellproliferation and MCT4 expression of LNCaP human prostate cancer cells48 hours after transfection: A) shows MCT4 ASOs are able to inhibitLNCaP cell proliferation to levels comparable to those observed withother human prostate cancer cell lines; and B) shows that a decrease incell proliferation is associated with a decrease in MCT4 expression,which suggests that the inhibitory effect of MCT4 ASOs may be moreassociated with a glycolytic phenotype than with androgen receptorstatus.

FIG. 8 shows that candidate MCT4 ASOs are able to inhibit PC-3 cellmigration and tissue invasion in vitro; A) shows treatment of PC3 cellswith candidate MCT4 ASOs resulted in an inhibition of cell migrationthrough a transwell, as indicated by a reduction in the number ofmigrated cells observed; and B) shows that the treatment with MCT4 ASOalso inhibited the ability of PC-3 cells to invade Matrigel, andsuggests that MCT4-mediated lactic acid secretion could play animportant role in cancer metastasis.

FIG. 9 shows that the treatment of nude mice with MCT4 ASO did not causehost toxicity as measured by animal weights: A) shows a plot of animalweights throughout the duration of the in vivo study, and treatment withMCT4 ASOs did not significantly affect the average animal weight of eachgroup; and B) shows that the individual animal weights also remainedstable throughout the treatment period.

FIG. 10 shows a comparison the total live cell numbers assessed 48 hoursafter treatment with SEQ ID NO:5 ASO, Metformin, Doxycycline, Docetaxeland MDV3100, wherein SEQ ID NO:5 ASO was tested in comparison to each ofMetformin, Doxycycline, Docetaxel and MDV3100, alone and in combination:(A) shows the total live PC3 cell numbers for SEQ ID NO:5 ASO andMetformin; (B) shows the total live PC3 cell numbers for SEQ ID NO:5 ASOand Doxycycline; (C) shows the total live PC3 cell numbers for SEQ IDNO:5 ASO and Docetaxel; and (D) shows the total live C4-2 cell numbersfor SEQ ID NO:5 ASO and MDV3100.

DETAILED DESCRIPTION

Any terms not directly defined herein shall be understood to have themeanings commonly associated with them as understood within the presentfield of art. Certain terms are discussed below, or elsewhere in thespecification, to provide additional guidance to the practitioner indescribing the compositions, devices, methods and the like ofembodiments, and how to make or use them. It will be appreciated thatthe same thing may be said in more than one way. Consequently,alternative language and synonyms may be used for any one or more of theterms discussed herein. No significance is to be placed upon whether ornot a term is elaborated or discussed herein. Some synonyms orsubstitutable methods, materials and the like are provided. Recital ofone or a few synonyms or equivalents does not exclude use of othersynonyms or equivalents, unless it is explicitly stated. Use of examplesin the specification, including examples of terms, is for illustrativepurposes only and does not limit the scope and meaning of theembodiments described herein.

A method is provided for “treating” a cancer cell, wherein treating ismeant to encompass preventing proliferation of the cell, amelioratingsymptoms associated with the cancer, and eradicating the cancer cell.The term “treating” as used herein is also meant to include theadministration at any stage of the cancer, including earlyadministration of a compound or late administration. A person of skillin the art would appreciate that the term “ameliorating” is meant toinclude the prospect of making a cancer more tolerable for a subjectafflicted therewith (for example, by reducing tumour load). A person ofskill in the art would also appreciate that the term “eradication” withregards to cancer would include elimination of the cancer cells in wholeor in part from a subject. Accordingly, as used herein “treatment” mayrefer to the prevention of cancer cell proliferation in a subject, theamelioration of symptoms associated with the cancer, the eradication ofthe cancer from a subject, or combinations thereof.

Antisense oligonucleotide compounds are typically single stranded RNAcompounds which bind to complementary RNA compounds, such as target mRNAmolecules, and block translation from the complementary RNA compounds bysterically interfering with the normal translational machinery. Thisprocess is usually passive, in that it does not require or involveadditional enzymes to mediate the RNA interference process. Specifictargeting of antisense RNA compounds to inhibit the expression of adesired gene may generally involve designing the antisense RNA compoundto have a homologous, complementary sequence to the desired gene.Perfect homology is not necessary for the RNA interference effect. Inone embodiment of the invention, the antisense RNA compounds include anyRNA compound with sufficient complementary homology to bind to the MCT4mRNA transcript causing a reduction in translation of the MCT4 protein.In another embodiment of the invention, the antisense RNA compoundsinclude any RNA compound with sufficient complementary homology to bindto the MCT4 mRNA transcript causing a reduction in translation of theMCT4 protein. The antisense compounds may be modified to enhance thestability of the oligonucleotides, particularly for in vivo use.Numerous examples of methods for designing and optimizing antisense RNAcompounds are found in the journal literature (i.e. Pan and Clawson2006; Patzel 2007; Peek and Behlke 2007). Perfect sequencecomplementarity is not necessary for the antisense compound to modulateexpression of the target gene. The present inventors providenon-limiting examples of antisense compounds which modulate theexpression of MCT4.

Antisense oligonucleotide sequences as described herein (see TABLES Aand B) or for use as described herein may be administered by means of amedical device or appliance such as an implant, graft, prosthesis,stent, etc. Also, implants may be devised which are intended to containand release such compounds or compositions. An example would be animplant made of a polymeric material adapted to release the compoundover a period of time.

The percent inhibition for all tested MCT4 ASOs is shown below in TABLEA. Generally a 50% inhibition was considered active for MCT4 ASOs.

TABLE A MCT₄ ASO Sequences and Percent Inhibition of MCT₄. SEQNucleotide % MCT4 Previous ID No. Sequence (5′ to 3′) Range InhibitionIdentifier  1 TCCCATGGCCAGGAGGGTTG 137-156 65.52 MCT₄ ASO #1  2GTCCCGGAAGACGCTCAGGT 806-825 86.33 MCT₄ ASO #3  3 AAGGACGCAGCCACCATGCC451-470 71.19 MCT₄ ASO #12  4 TTGGCGTAGCTCACCACGAA 892-911 77.13MCT₄ ASO #13  5 AGATGCAGAAGACCACGAGG 1110-1129 78.16 MCT₄ ASO #14  6CCACTCTGGAATGACACGGT 1719-1738 78.67 MCT₄ ASO #20  7GTAGGAGAAGCCAGTGATGAC 238-257 66.31 MCT₄ ASO #21  8 AGCATGGCCAGCAGGATGGA343-362 80.77 MCT₄ ASO #22  9 GGCTGGAAGTTGAGTGCCAA 526-545 76.03MCT₄ ASO #23 10 CATGCCGTAGGAGATGCCAA 1133-1152 76.51 MCT₄ ASO #24 11CTCAGGCTGTGGCTCTTTGG 1391-1410 63.01 MCT₄ ASO #27 12 TAGCGGTTCAGCATGATGA551-569 70.36 MCT₄ siRNA ASO #3 13 AGCACGGCCCAGCCCCAGCC 205-224 66.85MCT₄ + 1 ASO #1 14 GAGCTCCTTGAAGAAGACACT 277-296 72.00 MCT₄ + 1 ASO #215 CAGGATGGAGGAGATCCAGG 332-351 75.58 MCT₄ + 1 ASO #3 16AGACCCCCCACAAGCATGAC 418-437 55.47 MCT₄ + 1 ASO #4 17GAAGTTGAGTGCCAAACCCAA 520-539 70.93 MCT₄ + 1 ASO #5 18CCCGTTGGCCATGGGGCGCC 581-600 71.14 MCT₄ + 1 ASO #6 19GCCAGCCCGTTGGCCATGGG 586-605 80.26 MCT₄ + 1 ASO #7 20AGGAAGACAGGGCTACCTGC 610-629 66.26 MCT₄ + 1 ASO #8 21GCACACAGGAAGACAGGGCT 616-635 60.36 MCT₄ + 1 ASO #9 22CAGGGCACACAGGAAGACAG 620-639 79.96 MCT₄ + 1 ASO #10 23CAGCAGTTGAGCAGCAGGCC 703-722 65.41 MCT₄ + 1 ASO #11 24GACCTGTCCCGTAGAGCATG 357-396 47-24 MCT₄ ASO #2 25 TTCCCAAGCCCCGCCACGAA 997-1016 22.88 MCT₄ ASO #4 26 AATGCTCCACCTCCCGCAAG 1467-1486 9.55MCT₄ ASO #5 27 ACCTCCCCGTTTTTCTCAGG 1501-1520 3.17 MCT₄ ASO #6 28TGTGAACCACCTCCCCGTTT 1509-1528 29.91 MCT₄ ASO #7 29 TCTGTACCTCCTCCCTGTGC1570-1589 16.31 MCT₄ ASO #8 30 GAATGACACGGTTCCCACCC 1711-1730 25.51MCT₄ ASO #9 31 GCCCACCCACCCTCCCATTA 1870-1889 -43-4 MCT₄ ASO #10 32AAGAGACCCCCCACAAGCAT 421-440 18.67 MCT₄ ASO #11 33 CCCACCATGCCGTAGGAGAT1138-1157 49.00 MCT₄ ASO #15 34 AGTCCACCCCCGAGTCTGCA 1449-1468 20.5MCT₄ ASO #16 35 CTTCACCGCAGATCCACTCT 1732-1751 -10.07 MCT₄ ASO #17 36AACACTCCACCCACACGCAG 2028-2047 9,33 MCT₄ ASO #18 37 CCAGCCACTCAGACACTTGT1537-1556 33.14 MCT₄ ASO #19 38 GGCCACCGCCTCCATCAGCA 1229-1248 45.92MCT₄ ASO #25 39 CCTGAGCCAGTCCAGTTTGT 1616-1635 39.46 MCT₄ ASO #26 40CCCACCCACCCTCCCATTAA 1869-1888 -42.05 MCT₄ ASO #28 41GCTTCTGTACCTCCTCCCTG 1573-1592 17.02 MCT₄ ASO #29 42 TGTCGCTGTAGCCGATCCC310-328 21.20 MTC₄ siRNA ASO #1 43 TTAAAGTCACGTTGTCTCG 1854-1872 0.27MCT₄ siRNA ASO #2 44 TTGCGGCTTGGCTTCACCG 1744-1762 31.16MCT₄ siRNA ASO #4 45 CACAGCTCCTCCCATGGCCAGG 22.4 Suzuki Rat MCT₄ ASO 46ATGGCCAGGAGGGTTG 137-152 76.03 MCT₄ ASO #1.1 47 CATGGCCAGGAGGGTT 138-15381.21 MCT₄ ASO #1.2 48 CCATGGCCAGGAGGGT 139-154 77.65 MCT₄ ASO #1.3 49CCCATGGCCAGGAGGG 140-155 65.76 MCT₄ ASO #1.4 50 TCCCATGGCCAGGAGG 141-15630.52 MCT₄ ASO #1.5 51 CCTTCCCTGAAGGTTCCTCC 0.00 Control ASO

Antisense oligonucleotides (ASOs) that target MCT4 are provided herein.The use of MCT4 ASOs for administration to a cell is encompassed by themethods described herein. Some of the ASOs as used in the Examplessection had modified internucleoside linkages. In particular, the ASOshad phosphorothioate linkages between all nucleosides. MCT4 ASOs are 16to 20-mer oligonucleotides with modified phosphorothioate linkages.However, variations of MCT4 ASOs may also have unmodified phosphodiesterlinkages or partially modified linkages (i.e. any integer between 1 and19 phosphorothioate linkage(s) or other modified linkages). Alternativemodifications are also known in the art.

Antisense oligonucleotides (ASOs) of 16 to 20-mer in length that targetMCT4 are provided herein. However, it would be apparent to a personskilled in the art that biologically active oligonucleotide comprisingthe 16, 19 or 20 nucleotides of SEQ ID NO: 1-23 or SEQ ID NO. 46-49 mayalso be used.

A phosphorothioate oligonucleotide bond modification alters thephosphate linkage by replacing one of the non-bridging oxygens withsulfur. The introduction of phosphorothioate linkages alters thechemical properties of the oligonucleotide. In particular, the additionof phosphorothioate linkages reduces nuclease degradation of theoligonucleotide and thereby increasing the half-life in situ.Accordingly, this modification is particularly useful for antisenseoligonucleotides, which when introduced into cells or biologicalmatrices can interact with target nucleic acids to silence theexpression of a particular transcript. Oligonucleotides containingphosphorothioate linkages accomplish this feat either through directblockage of translation or enable enzymatic degradation of the targettranscript (for example, via RNase H).

Although phosphorothioate linkages provide improved half-life, theintroduction of these linkages into an oligonucleotide may alsointroduce limitations to their function as antisense oligonucleotides.Each phosphorothioate linkage creates a chiral center at each bond,which may result in multiple isomers of the oligonucleotide generatedduring synthesis and the isomers may have differential characteristicsand functional properties. However much of the isomer effects may bemitigated through careful positioning of the modifications or by usingadditional modifications in conjunction with the phosphorothioate bonds.

One or more of the phosphorothiodiester linkages of the oligonucleotidemoiety may be modified by replacing one or both of the two bridgingoxygen atoms of the linkage with analogues such as —NH, —CH2, or —S.Other oxygen analogues known in the art may also be used.

A “modified oligonucleotide” as used herein is meant to includeoligonucleotides that are substituted or modified. In addition to thenaturally occurring primary bases adenine, guanine, cytosine, andthymine, or other natural bases such as inosine, deoxyinosine, andhypoxanthine, there are numerous other modifications. For example,isosteric purine 2′ deoxy-furanoside analogues, 2′-deoxynebularine or 2′deoxyxanthosine, or other purine and pyrimidine analogues such as5-methyl pyrimidine or a 5-propynyl pyrimidine may also be utilized toimprove stability and target hybridization.

A “modified sugar” as used herein when discussing an oligonucleotidemoiety, a sugar modified or replaced so as to be ribose, glucose,sucrose, or galactose, or any other sugar. Alternatively, theoligonucleotide may have one or more of its sugars substituted ormodified in its 2′ position, i.e. 2′ alkyl or 2′-o-alkyl. An example ofa 2′-O-allyl sugar is a 2′-O-methylribonucleotide. Furthermore, theoligonucleotide may have one or more of its sugars substituted ormodified to form an -anomeric sugar.

“Second-generation” oligonucleotides as used herein mat be defined asoligonucleotides that are resistant to degradation by cellular nucleasesand capable of hybridizing specifically to their target mRNA with equalor higher affinity than first generation ASOs. An example of a 2^(nd)generation ASO is a 2′-O-(2-Methoxyethyl)-RNA (2′MOE gapmermodification). With a 2′-MOE gapmer the 5′ and 3′ ends may have 2′-MOEmodified nucleotides to protect against degradation, but the gap betweenthe 5′ and 3′ ends may be unmodified phosphodiester linkages. Numerousother chemical modifications have been developed to improve ASOs. Forexample, morpholino, N3′ to P5′ phosphoramidate, and methylphosphonatechemical modifications are known in the art (N. Dias, and C. A. Stein2002). Furthermore, peptide nucleic acids (PNAs) may also be used.

An alignment of tested 16 mer truncations of SEQ ID NO:1 are shown inTABLE B along with their % MCT4 inhibitions,

Alignment of SEQ ID NO 1 with 16 mer Truncations SEQ % MCT4 ID NOSEQUENCE Inhibition  1 TCCCATGGCCAGGAGGGTTG 65.52 46 ATGGCCAGGAGGGTTG76.03 47 CATGGCCAGGAGGGTT 81.21 48 CCATGGCCAGGAGGGT 77.65 49CCCATGGCCAGGAGGG 65.76 50 TCCCATGGCCAGGAGG 30.52

The compounds, as described herein, may be in isolation, or may belinked to or in combination with tracer compounds, liposomes,carbohydrate carriers, polymeric carriers or other agents or excipientsas will be apparent to one of skill in the art. In alternateembodiments, such compounds may further comprise an additionalmedicament, wherein such compounds may be present in a pharmacologicallyeffective amount.

The term “medicament” as used herein refers to a composition that may beadministered to a patient or test subject and is capable of producing aneffect in the patient or test subject. The effect may be chemical,biological or physical, and the patient or test subject may be human, ora non-human animal, such as a rodent (for example, a transgenic mouse, amouse or a rat), dog, cat, cow, sheep, horse, hamster, guinea pig,rabbit or pig. The medicament may be comprised of the effective chemicalentity alone or in combination with a pharmaceutically acceptableexcipient.

The term “pharmaceutically acceptable excipient” may include any and allsolvents, dispersion media, coatings, antibacterial, antimicrobial orantifungal agents, isotonic and absorption delaying agents, and the likethat are physiologically compatible. An excipient may be suitable forintravenous, intraperitoneal, intramuscular, subcutaneous, intrathecal,topical or oral administration. An excipient may include sterile aqueoussolutions or dispersions for extemporaneous preparation of sterileinjectable solutions or dispersion. Use of such media for preparation ofmedicaments is known in the art.

Compositions or compounds according to some embodiments described hereinmay be administered in any of a variety of known routes. Examples ofmethods that may be suitable for the administration of a compoundinclude orally, intravenous, inhalation, intramuscular, subcutaneous,topical, intraperitoneal, intra-rectal or intra-vaginal suppository,sublingual, and the like. The compounds described herein may beadministered as a sterile aqueous solution, or may be administered in afat-soluble excipient, or in another solution, suspension, patch, tabletor paste format as is appropriate. A composition comprising thecompounds described herein may be formulated for administration byinhalation. For instance, a compound may be combined with an excipientto allow dispersion in an aerosol. Examples of inhalation formulationswill be known to those skilled in the art. Other agents may be includedin combination with the compounds described herein to aid uptake ormetabolism, or delay dispersion within the host, such as in acontrolled-release formulation. Examples of controlled releaseformulations will be known to those of skill in the art, and may includemicroencapsulation, embolism within a carbohydrate or polymer matrix,and the like. Other methods known in the art for making formulations arefound in, for example, “Remington's Pharmaceutical Sciences”, (19thedition), ed. A. Gennaro, 1995, Mack Publishing Company, Easton, Pa.

The dosage of the compositions or compounds of some embodimentsdescribed herein may vary depending on the route of administration(oral, intravenous, inhalation, or the like) and the form in which thecomposition or compound is administered (solution, controlled release orthe like). Determination of appropriate dosages is within the ability ofone of skill in the art. As used herein, an “effective amount”, a“therapeutically effective amount”, or a “pharmacologically effectiveamount” of a compound refers to an amount of an ASO as described hereinpresent in such a concentration to result in a therapeutic level of thecompound delivered over the term that the compound is used. This may bedependent on the mode of delivery, time period of the dosage, age,weight, general health, sex and diet of the subject receiving thecompound. Methods of determining effective amounts are known in the art.It is understood that it could be potentially beneficial to restrictdelivery of the compounds described herein to the target tissue or cellin which inhibition of MCT4 expression is desired. It is also understoodthat it may be desirable to target the compounds described herein to adesired tissue or cell type. The compounds described herein may thus becoupled to a targeting moiety. The compounds may be coupled to a celluptake moiety. The targeting moiety may also function as the cell uptakemoiety.

In general, antisense oligonucleotides as described herein may be usedwithout causing substantial toxicity. Toxicity of the compounds asdescribed herein can be determined using standard techniques, forexample, by testing in cell cultures or experimental animals anddetermining the therapeutic index, i.e., the ratio between the LD50 (thedose lethal to 50% of the population) and the LD100 (the dose lethal to100% of the population). In some circumstances however, such as insevere disease conditions, it may be appropriate to administersubstantial excesses of the compositions. Some antisenseoligonucleotides as described herein may be toxic at someconcentrations. Titration studies may be used to determine toxic and nontoxic concentrations. Toxicity may be evaluated by examining aparticular antisense oligonucleotide's specificity across cell lines.Animal studies may be used to provide an indication if the compound hasany effects on other tissues.

In some embodiments, at least one antisense oligonucleotide as describedherein may be used, for example, and without limitation, for treating acancer cell. As used herein “at least one” is intended to mean one ormore, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27 etc.

In some embodiments, antisense oligonucleotides as described herein maybe used, for example, and without limitation, in combination with othertreatment methods for at least one indication selected from malignanciesin which elevated expression of MCT4 is observed. These include, but arenot limited to, prostate cancer; renal cell carcinoma; breast cancer;cervical cancer; liver cancer; bladder cancer; and small cell lungcancer in mammals, including humans. Antisense oligonucleotides and maybe used as neoadjuvant (prior), adjunctive (during), and/or adjuvant(after) therapy with surgery, radiation (brachytherapy or externalbeam), or other therapies (for example, HIFU). For example, MCT4antisense oligonucleotides as described herein may be used incombination with docetaxel, MDV3100, or with modulators of glucosemetabolism (including doxycycline or other mitochondrial inhibitors).

Methods and Materials

The following methods and materials were employed with respect to theEXAMPLES described herein.

Cell Cultures

Human PC-3 and DU145 CRPC cells, human LNCaP prostate cancer cells, andmouse TRAM PC2 prostate cancer cells were purchased from the AmericanType Culture Collection (ATCC); C4-2 CRPC cells were from the VancouverProstate Centre, Vancouver, BC, Canada. Human monolayer cultures weremaintained in RPMI-1640 (GE Healthcare Hyclone™, Logan, Utah)supplemented with 10% fetal bovine serum (FBS) (GE Healthcare Hyclone™,Logan, Utah) while TRAMPC2 cells were maintained in DMEM (GE HealthcareHyclone™, Logan, Utah) supplemented with 5% FBS. For cell counting,cells were trypsinized to form a single cell suspension and countedusing a TC20 Automated Cell Counter (Bio-Rad, Hercules, Calif.). Cellviability was assessed by trypan blue exclusion.

Human Prostate Cancer Tissue Microarray (TMA) Construction andImmunohistochemistry

TMAs were manually constructed, as previously described (Chiang et al.,2014; Thomas et al., 2011), using various Gleason grades-exhibitingprostate cancer specimens (n=342), obtained from the Vancouver ProstateCentre Tissue Bank with written informed patients' consent, clinicalinformation and institutional study approval. All specimens wereobtained through radical prostatectomy, except CRPC samples that wereobtained via transurethral resection of the prostate (TURP).Immunohistochemical staining was conducted using a Ventana™ autostainer(model Discover XT™; Ventana Medical System™, Tucson, Ariz.) with anenzyme-labelled biotin-streptavidin system and a solvent-resistant DABMap kit (Ventana™). Staining intensity was scored by a trainedpathologist on a four-point scale: 0 represents no staining on any tumorcells, 1 represents a faint or focal, questionably present stain, 2represents a stain of convincing intensity in a minority of cells, and 3represents a stain of convincing intensity in a majority of cells.

Antibodies

The following antibodies and conjugates were used: rabbit anti-MCT4antibody (Santa Cruz™, Santa Cruz, Calif.; WB 1:4000, IHC 1:100), mouseanti-vinculin antibody (Sigma™; WB 1:1000), rabbit anti-cleaved caspase3 antibody (Cell Signalling Technology™, Danvers, Mass.; IHC 1:50),mouse anti-Ki67 antibody (Dako™, Burlington, ON; IHC 1:50), ratanti-CD31 antibody (Dianova™, Hamburg, Germany; IHC 1:20), mouseanti-pan-T cell marker CD3 antibody (Dako™; IHC 1:50), biotinylatedmouse anti-NK1.1 (Cedarlane™, Burlington, ON; IHC 1:100), IRDye 800CWgoat anti-mouse antibody (Li-Cor Biosciences™, Lincoln, Nebr.; WB1:10,000), IRDye 680RD goat anti-rabbit antibody (Li-Cor Biosciences™;WB1:10,000), biotinylated goat anti-rabbit antibody (VectorLaboratories™, Burlingame, Calif.; IHC 1:200), biotinylated goatanti-rat antibody (Vector Laboratories™; IHC1:200), and biotinylatedgoat anti-mouse antibody (Vector Laboratories™; IHC 1:200).

ASO Design and Selection

First-generation phosphorothioate-modified ASOs against human MCT4 wererationally designed by selecting sequences containing favourable motifswhile excluding unfavourable ones (Matveeva et al., 2000). Specificityof MCT4-targeting sequences, compared to human and mouse genes (at least3 of 20 bases mismatched), was evaluated using BLAST. Ten sequences SEQID No: 1, 4, 5, 9, 11, 30, 36, 37, 40 and 41 (see TABLE A) distributedthroughout the length of the transcript with perfect complementarity toall human MCT4 transcript variants (NM_001042422.2, NM_001042423.2,NM_001206950.1, NM_001206951.1, NM_001206952.1, and NM_004207.3) wereselected and synthesized by Eurofins MWG Operon. The knock-downefficiencies of these ten ASOs were tested by determining target mRNAand protein expression 48 hours after transfection of cells using qPCRand Western blotting. Two candidate ASOs (#1 and #14—SEQ ID NOs: 1 and5, respectively) were selected for further studies. Sequences: ASO #1(SEQ ID NO: 1), 5′-TCCCATGGCCAGGAGGGTTG-3′; ASO #14 (SEQ ID NO: 5),5′-AGATGCAGAAGACCACGAGG-3′; a published non-targeting control ASO,5′-CCTTCCCTGAAGGTTCCTCC-3′ (Mullick et al., 2011; Samuel et al., 2006).

A person of skill in the art based on the general knowledge in the artand the information provided herein would be able to synthesize the ASOsdescribed herein or modify the ASOs described herein.

ASO and siRNA Transfection

Cells were transfected in 6-well plates with ASOs at 100 nM for 48 hours(unless otherwise indicated) using Oligofectamine™ (Invitrogen™,Carlsbad, Calif.) or with MCT4-targeting siRNAs and controls(Dharmacon™, Chicago, Ill.) at 50 nM for 48 hours using Lipofectamine™2000 (Invitrogen™), following the manufacturer's instructions.

Quantitative PCR

Total RNA was isolated using the RNeasy Mini Kit™ (Qiagen Inc.™, Hilden,Germany) and cDNA synthesized using the QuantiTect Reverse TranscriptionKit™ (Qiagen™) Primers were designed using Primer-BLAST™. qRT-PCRreactions using KAPA SYBR Fast Universal™ (Kapa Biosystems™, Woburn,Mass.) were performed in triplicate in a ViiA 7 Real-Time PCR™ system(Applied Biosystems™, Foster City, Calif.). Target genes were normalizedto a geometric average of 3 internal reference genes (Vandesompele etal., 2002).

Western Blotting

Cells were harvested and lysed in RIPA buffer (50 mM Tris-Cl pH 7.4, 150mM NaCl, 1% Igepal, 0.5% Na-deoxycholate, 0.1% SDS) supplemented with acomplete protease inhibitor cocktail (Roche, Nutley, N.J.). The proteinconcentration of the lysate was determined by Pierce BCA Protein Assay™(Thermo Scientific™, Waltham, Mass.). The lysate was run on 8% SDSpolyacrylamide gel (20 ug of protein per lane) and proteins weretransferred onto PVDF membrane (Millipore™, Billerica, Mass.). The blotwas blocked with the Odyssey™ blocking buffer (Li-cor Biosciences™) andprobed with anti-MCT4 antibody. Vinculin was used as a loading control.Following overnight incubation at 4° C., the primary antibody was probedwith the corresponding secondary antibody and detected using the OdysseyInfrared Imaging System™ (Li-cor Biosciences™) and Image Studio Version3.1™ (Li-cor Biosciences™) Densitometry analysis was done using ImageJ(U. S. National Institutes of Health, Bethesda, Md.).

Modified Boyden Chamber Assay

The migration and invasion potential of PC-3 cells following treatmentwith MCT4 ASOs was investigated using Matrigel-coated modified Boydenchambers (BD Bioscience™, San Jose, Calif.) as previously described(Chiang et al., 2014). Briefly, ASO-treated cells were seeded into thetop chamber at 50,000 live cells per well. The cells were thenre-suspended after 48 hours using dissociation buffer (Trevigen™,Gaithersburg, Md.) containing calcein AMS (12.5 mM; Trevigen™). Thenumber of migrated/invaded cells in the lower chamber was determined byfluorescence measurement (485 nm excitation, 520 nm emission) of thecell suspensions using the Infinite F500 Fluorometer™ (Tecan™,Männedorf, Switzerland).

Lactate and Glucose Determination

PC-3 cells transfected with ASOs for 48 hours were assessed for lactateand glucose levels. Cells were incubated with fresh media for 4 hours. Asample of the media was then taken and deproteinated with 10K SpinColumns™ (BioVision™, Milpitas, Calif.) prior to determination oflactate concentration using Lactate Assay Kit (BioVision) and glucoseconcentration using Glucose Assay Kit™ (BioVision™). Intracellularlactate levels were determined by lysing ASO-transfected cells in MQH2O.Final concentrations were determined by normalizing to the total numberof live cells.

Treatment with MCT4 ASO of PC-3 Tumor-Bearing Nude Mice

PC-3 cells (106 cells in 1:1 HBSS:Matrigel) were injected subcutaneouslyinto both flanks of 24 male athymic nude mice (Simonsen Laboratories™,Gilroy, Calif.). Once the mean tumour volume had reached approximately100 mm3, mice were randomized into four groups and treated withintraperitoneal injections of MCT4 ASO #1 (SEQ ID NO: 1), #14 (SEQ IDNO: 5), control ASO, or vehicle (PBS) at 10 mg/kg daily for 5 daysfollowed by 2 days off treatment for a total of 15 days. The health ofthe mice was monitored throughout the study by measuring body weightsand checking for abnormal behaviour such as lethargy, lack of hydration,and additional signs of weakness. Tumour size was measured twice weeklyand tumour volume calculated using the formula:Volume=Length×width×depth×0.5236 (mm3). Mice were sacrificed 1 hourafter the final dose for tissue harvesting.

Immunohistochemistry of Tumour Tissue

Tumour tissue was formalin-fixed and paraffin-embedded forimmunohistochemical analysis. Tissues were sectioned, probed, andstained with DAB (Sigma™) as previously described (Wang et al., 2005).For Ki-67 and cleaved caspase 3 staining, images of five random fieldsat 400× magnification were taken per tumour and cells counted todetermine the percentage of positively stained cells. For MCT4, imagesof five random fields at 200× magnification were taken per tumour andstaining intensity was assessed by percentage scoring, using theformula: Intensity=(% area score 3)×3+(% area score 2)×2+(% area score1)×1. The extent of immune cell aggregation was quantified followingCD31 staining by imaging the five most prominent regions of aggregatesper tumour at 200× magnification and determining the percent area of thefield they occupied. The proportions of immune cells were evaluated asthe area of positive staining normalized to the area occupied by immunecell aggregates in the same five prominent regions.

Statistical Analysis

All pooled results are represented as Mean±SEM. Statistical analysis wasperformed using GraphPad Prism 6™ (GraphPad Software, Inc., La Jolla,Calif.). The Student t-test was carried out to compare means between twogroups. One-way ANOVA followed by the post-hoc Dunnett's test was usedto compare means of more than two groups. Two-way ANOVA followed bypost-hoc multiple comparison was applied to compare tumour growth. Acontingency test was done to compare staining intensity between patientcohorts on the tissue microarray. A Log-rank test was done to comparepatient survival curves. Chi-squared tests were done to correlate MCT4expression levels with various clinical parameters. Results with ap-value<0.05 were considered statistically significant and are indicatedby * for p<0.05, ** for p<0.01, and *** for p<0.001.

EXAMPLES Example 1: Elevated MCT4 Protein Expression is Associated withCRPC

A tissue microarray (TMA) composed of tissues from Gleason grade 3, 4and 5 human prostate cancers was stained for MCT4 protein. As shown inFIGS. 1A and B, Gleason grade 5 prostate cancers had significantlyincreased MCT4 protein expression relative to Gleason grade 3 and 4specimens. Also, elevated MCT4 expression was associated with an earliertime to relapse from primary treatment as measured by increases in serumPSA levels, with the high MCT4-expressing cohort having a median time torelapse of 63.3 months vs. 94.2 months for the low MCT4-expressingcohort (FIG. 1C). Additionally, high MCT4 expression was correlated withother clinical characteristics associated with poor prognosis, such ashigher serum PSA levels at diagnosis and clinical T stage (TABLE C).Furthermore, elevated MCT4 protein expression was found in tumours frompatients subjected to prolonged neo-adjuvant hormone therapy (>6 months)and CRPC patients (FIGS. 1D and E), indicating that elevated expressionof MCT4 protein in prostate cancer is associated with development ofCRPC.

TABLE C Clinico-pathological Characteristics Associated with High MCT4Expression in Patient Samples Low MCT4 (0 + 1) High MCT4 (2 + 3) p-valueGleason <7 60.7% (17/28) 39.3% (11/28) 0.0061**  7 52.0% (53/102) 48.0%(49/102) >7 29.1% (16/55) 70.9% (39/55) PSA ≤10 ng/mL 56.3% (71/126)43.7% (55/126) 0.0157*  >10 ng/mL 37.3% (22/59) 62.7% (37/59) T Stage≤pT2 55.4% (51/92) 44.6% (41/92) 0.0040** ≥pT3 34.4% (32/93) 65.6%(61/93) Surgical Margin Negative 48.5% (50/103) 51.5% (53/103) 0.2595Positive 40.2% (33/82) 59.8% (49/82) Capsule Invasion Negative 52.1%(49/94) 47.9% (45/94) 0.0435* Positive 37.4% (34/91) 62.6% (57/91) SVInvasion Negative 48.3% (72/149) 51.7% (77/149) 0.0544 Positive 30.6%(11/36) 69.4% (25/36)

Example 2: Knockdown of MCT4 Inhibits PC-3 Cell Proliferation

The potential therapeutic efficacy of inhibiting MCT4 expression wasinvestigated using MCT4-targeting siRNAs and ASOs. Human PC-3 CRPC cellswere used as they present a distinct glycolytic metabolic profile, aproperty associated with MCT4 expression (Vaz et al., 2012). As shown inFIG. 2A, treatment of PC-3 cells with MCT4 siRNA led to an inhibition ofcell proliferation, suggesting potential therapeutic efficacy of an MCT4knockdown approach. Accordingly, ASOs specifically targeting human MCT4were designed. Screening of ten MCT4 ASOs revealed varying capacities ofinhibiting PC-3 cell proliferation and MCT4 expression, with ASOs #1(SEQ ID NO: 1) and #14 (SEQ ID NO: 5) showing the greatest potency(FIGS. 2B and 2C). A strong correlation was found between the reducedlevels of MCT4 mRNA in PC-3 cells treated with the various ASOs and theresulting cell numbers (FIG. 2D), indicating that the growth inhibitionby the ASOs was directly related to MCT4 knockdown.

Example 3: MCT4 ASOs Inhibit PC-3 Cell Proliferation

The growth-inhibitory activities of MCT4 ASOs #1 (SEQ ID NO: 1) and #14(SEQ ID NO: 5) were further characterized. As shown in FIG. 2E, PC-3cell proliferation was inhibited by both ASOs in a dose-dependentmanner, with ASO #14 being slightly more effective (IC50=26 nM) than ASO#1 (IC50=50 nM). The ASOs also reduced MCT4 mRNA levels in adose-dependent manner, mirroring their inhibition of cell proliferation(IC50 ASO #14=32 nM, IC50 ASO #1=50 nM). As shown in FIG. 2F, both ASOsmaintained the inhibition of cell proliferation even at 96 hourspost-transfection. While MCT4 mRNA levels began to increase slightlystarting after 48 hours of transfection, MCT4 protein levels remainedlow even at 96 hours.

Example 4: MCT4 ASOs Exert Growth-Inhibition and a Reduction in MCT4Expression in a Variety of Human Prostate Cancer Cell Lines and notMouse MCT4

When the MCT4 ASOs #1 (SEQ ID NO: 1) and #14 (SEQ ID NO: 5) weretransfected into human C4-2 CRPC cells they inhibited theirproliferation with IC50 values comparable to those observed with PC-3cells, i.e. ASO #1=40 nM, ASO #14=27 nM. Similarly, they reduced theMCT4 expression with IC50 values of 50 nM for ASO #1 and 26 nM for ASO#14 (FIG. 3A). Furthermore, when the ASOs were transfected into humanDU145 prostate cancer cells, a similar inhibitory effect on cellproliferation and MCT4 expression was observed with almost identicalIC50 values (FIG. 3B). Transfection of ASOs into LNCaP prostate cancercells also showed a similar inhibition of cell proliferation and MCT4expression (FIG. 7), suggesting that the inhibitory effect is moreassociated with a glycolytic phenotype than androgen receptor status.Importantly, transfection of MCT4 ASOs #1 and #14 into mouse TRAMPC2prostate cancer cells did not lead to a significant reduction in cellproliferation or mouse MCT4 expression (FIG. 3C). Taken together, theresults suggest that these ASOs specifically target human MCT4 and thattheir inhibitory effect on cell proliferation is a consequence of MCT4knock-down.

Example 5: Candidate MCT4 ASOs Inhibit Glucose Metabolism and TissueInvasion/Migration of CRPC Cells In Vitro

To further examine the effects of MCT4-targeting ASOs on prostate cancercells, we measured their effects on lactic acid secretion, intracellularlactate concentrations, and glucose consumption of PC-3 cells.Transfection of the cells with MCT4 ASOs #1 (SEQ ID NO: 1) and #14 (SEQID NO: 5) led to a marked inhibition of lactic acid secretion, acorresponding accumulation of intracellular lactate and an extensivedecrease in glucose consumption, measured after 48 hours of transfection(FIG. 4A). Furthermore, as shown in FIG. 4B, treatment with the ASOsresulted in down regulation of various genes involved in glycolysis,i.e. GAPDH, PGK1, PGAM1 and ENO1. In addition, expression of lactatedehydrogenase A (LDHA) was found to be depressed, indicative of adecrease in the conversion of pyruvate to lactic acid. Moreover,decreased expression was found for pyruvate dehydrogenase kinase-1(PDK1), an enzyme that shunts pyruvate away from the TCA cycle andpromotes its conversion to lactic acid. Thus the treatment with the MCT4ASOs led to inhibition of aerobic glycolysis.

Treatment with MCT4 ASOs also inhibited the migration and tissueinvasion of PC-3 cells in modified Boyden chambers (FIG. 8), suggestingthat lactic acid secretion as facilitated by MCT4 could also play animportant role in the metastatic process.

Example 6: Growth of PC-3 Xenografts in Nude Mice Treated with MCT4 ASOs

Male athymic nude mice bearing subcutaneous PC-3 tumours were treatedwith MCT4 ASOs #1 (SEQ ID NO: 1) and #14 (SEQ ID NO: 5) for a total of15 days. Both ASOs markedly inhibited the growth of the tumours (FIG.5A) without inducing major host toxicity as assessed by monitoringanimal weights (FIG. 9) and behaviour. Immunohistochemical analysisrevealed that the ASO-induced inhibition of tumour growth was associatedwith an increase in cell apoptosis, as measured by cleaved-caspase 3staining, and a decrease in cell proliferation, as measured by Ki-67staining (FIG. 5B). The decrease in tumour growth was associated with adecrease in MCT4 protein expression (FIG. 5C), consistent with ananti-proliferative effect generated by MCT4 knockdown. Representativeimages of tumours from each group showed presence of strong membranestaining in the control tumours, which was absent in the MCT4ASO-treated tumours (micrographs not shown).

Example 7: Effects of MCT4 ASOs on Immune Cell Aggregates in Nude Mice

As lactic acid-induced acidification of tumours has been linked tosuppression of local host anticancer immunity (Choi et al., 2013), it isof interest to determine whether the treatment of the PC-3tumour-bearing nude mice with MCT4 ASOs causes changes in the local hostimmune response of these mice even though their immune reactivity wasvery limited. To that end, immune cell aggregates that had extravasatedfrom CD31-positive blood vessels, particularly in the tumour periphery,were quantified. As shown in FIG. 6A, xenografts treated with the twoMCT4 ASOs had significantly larger immune cell aggregates compared tocontrol tumours. When micrographs were examined they showed immuneaggregates are characterized by areas of small, circular, densely packednuclei that are distinct from the surrounding tumour cells and stainingfor CD31 reveals that these immune cells have extravasated and surroundthe blood vessels in the tumour periphery (micrographs not shown).Quantification of the natural killer (NK) cell population, thepredominant cytotoxic immune cell subtype in nude mice (Shultz et al.,2007), revealed that the treatment with the MCT4 ASOs markedly increasedthe proportion of tumour-associated NK cells (FIG. 6B). Furthermore,activation of NK cells is facilitated by CD3 (Koch et al., 2013), amolecule commonly regarded as a T-cell marker for its association withthe T-cell receptor complex. Its expression in NK cells is detectable byimmunohistochemistry (Morice, 2007), and in view of the absence of Tcells in nude mice, can be used as an indicator of NK cell activation(Lanier et al., 1992). Treatment with MCT4 ASO also significantlyaltered the composition of the immune cells present in the aggregates,whereby staining using the NK cell marker NK1.1 revealed that there wasan increased proportion of NK cells associated with the tumour(micrographs not shown). As shown in FIG. 6C, the proportion of CD3positive cells increased with MCT4 ASO treatment. While stainingrevealed that the proportion of activated NK cells associated with theASO-treated tumours had also increased, suggesting stimulation ofanti-cancer immunity.

Example 8: Combination Therapies with SEQ ID NO: 5

Various combination strategies were screened at the approximate IC₅₀concentrations to determine whether synergistic effects may be found bycombining current cancer therapeutics (see TABLE D) with MCT4 ASO SEQ IDNO:5. In FIGS. 10 A-C, the total live PC3 cell numbers were assessed 48hours after treatment with Metformin, Doxycycline and docetaxel bothalone and in combination with MCT4 ASO SEQ ID NO:5. However, C4-2 cellswere used to test MDV3100, SEQ ID NO:5 and a combination of SEQ ID NO:5and MDV3100 (FIG. 10D). The combination of Docetaxel with MCT4 ASO SEQID NO:5 showed synergistic effect with Combination Index=0.780.

TABLE D IC₅₀ Concentrations of SEQ ID No. 5, Metformin, Doxycycline,Docetaxel MDV3100 IC₅₀ Drug Concentration Drug Category SEQ ID No. 5 50nM MCT4 inhibitory ASO Metformin 4 mM Modulator of glucose metabolismDoxycycline 15 μM Possible mitochondrial inhibitor Docetaxel 2.5 nMApproved chemotherapy for prostate cancer MDV3100 50 μM Approvedsecond-generation anti- androgen for prostate cancer

Although embodiments described herein have been described in some detailby way of illustration and example for the purposes of clarity ofunderstanding, it will be readily apparent to those of skill in the artin light of the teachings described herein that changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims. Such modifications include thesubstitution of known equivalents for any aspect of the invention inorder to achieve the same result in substantially the same way. Numericranges are inclusive of the numbers defining the range. The word“comprising” is used herein as an open ended term, substantiallyequivalent to the phrase “including, but not limited to”, and the word“comprises” has a corresponding meaning. As used herein, the singularforms “a”, “an” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a thing”includes more than one such thing. Citation of references herein is notan admission that such references are prior art to an embodiment of thepresent invention. The invention includes all embodiments and variationssubstantially as herein described and with reference to the figures.

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What is claimed is:
 1. An antisense oligonucleotide (ASO), wherein theASO has a sequence selected from the following: (SEQ ID NO: 1) (a)TCCCATGGCCAGGAGGGTTG; (SEQ ID NO: 2) (b) GTCCCGGAAGACGCTCAGGT;(SEQ ID NO: 3) (c) AAGGACGCAGCCACCATGCC; (SEQ ID NO: 4) (d)TTGGCGTAGCTCACCACGAA; (SEQ ID NO: 5) (e) AGATGCAGAAGACCACGAGG;(SEQ ID NO: 6) (f) CCACTCTGGAATGACACGGT; (SEQ ID NO: 7) (g)GTAGGAGAAGCCAGTGATGAC; (SEQ ID NO: 8) (h) AGCATGGCCAGCAGGATGGA;(SEQ ID NO: 9) (i) GGCTGGAAGTTGAGTGCCAA; (SEQ ID NO: 10) (j)CATGCCGTAGGAGATGCCAA; (SEQ ID NO: 11) (k) CTCAGGCTGTGGCTCTTTGG;(SEQ ID NO: 13) (l) AGCACGGCCCAGCCCCAGCC; (SEQ ID NO: 14) (m)GAGCTCCTTGAAGAAGACACT; (SEQ ID NO: 15) (n) CAGGATGGAGGAGATCCAGG;(SEQ ID NO: 16) (o) AGACCCCCCACAAGCATGAC; (SEQ ID NO: 17) (p)GAAGTTGAGTGCCAAACCCAA; (SEQ ID NO: 18) (q) CCCGTTGGCCATGGGGCGCC;(SEQ ID NO: 19) (r) GCCAGCCCGTTGGCCATGGG; (SEQ ID NO: 20) (s)AGGAAGACAGGGCTACCTGC; (SEQ ID NO: 21) (t) GCACACAGGAAGACAGGGCT;(SEQ ID NO: 22) (u) CAGGGCACACAGGAAGACAG; (SEQ ID NO: 23) (v)CAGCAGTTGAGCAGCAGGCC; (SEQ ID NO: 46) (w) ATGGCCAGGAGGGTTG;(SEQ ID NO: 47) (x) CATGGCCAGGAGGGTT; (SEQ ID NO: 48) (y)CCATGGCCAGGAGGGT; and (SEQ ID NO: 49) (z) CCCATGGCCAGGAGGG;

and wherein the ASO comprises a modified internucleoside linkage, amodified sugar moiety, or modified nucleobase, and wherein the ASO isnot longer than 21 nucleotides in length.
 2. The ASO of claim 1, whereinthe ASO comprises a modified internucleoside linkage.
 3. The ASO ofclaim 2, wherein the modified internucleoside linkage is apeptide-nucleic acid linkage, a morpholino linkage, a N3′ to P5′phosphoramidate linkage, a methylphosphonate linkage or aphosphorothioate linkage.
 4. The ASO of claim 1, wherein the ASOcomprises a modified sugar moiety.
 5. The ASO of claim 4, wherein themodified sugar moiety is 2′-O-alkyl oligoribonucleotide.
 6. The ASO ofclaim 1, wherein the ASO has a 2′MOE gapmer modification.
 7. The ASO ofclaim 1, wherein the ASO comprises a modified nucleobase.
 8. The ASO ofclaim 7, wherein the modified nucleobase is a 5-methyl pyrimidine or a5-propynyl pyrimidine.
 9. A pharmaceutical composition, the compositioncomprising (a) the antisense oligonucleotide (ASO) of claim 1; and (b) apharmaceutically acceptable carrier.
 10. An antisense oligonucleotide(ASO), wherein the ASO has the sequence of TAGCGGTTCAGCATGATGA (SEQ IDNO:12), and wherein the ASO comprises a modified internucleosidelinkage, a modified sugar moiety, or modified nucleobase, and whereinthe ASO is single stranded and is not longer than 21 nucleotides inlength.
 11. The ASO of claim 10, wherein the ASO comprises a modifiedinternucleoside linkage.
 12. The ASO of claim 11, wherein the modifiedinternucleoside linkage is a peptide-nucleic acid linkage, a morpholinolinkage, a N3′ to P5′ phosphoramidate linkage, a methylphosphonatelinkage or a phosphorothioate linkage.
 13. The ASO of claim 10, whereinthe ASO comprises a modified sugar moiety.
 14. The ASO of claim 13,wherein the modified sugar moiety is 2′-O-alkyl oligoribonucleotide. 15.The ASO of claim 10, wherein the ASO has a 2′MOE gapmer modification.16. The ASO of claim 10, wherein the ASO comprises a modifiednucleobase.
 17. The ASO of claim 16, wherein the modified nucleobase isa 5-methyl pyrimidine or a 5-propynyl pyrimidine.
 18. A pharmaceuticalcomposition, the composition comprising (a) the antisenseoligonucleotide (ASO) of claim 10; and (b) a pharmaceutically acceptablecarrier.
 19. An antisense oligonucleotide (ASO), wherein the ASO has asequence selected from the following: (SEQ ID NO: 1)(a) TCCCATGGCCAGGAGGGTTG; (SEQ ID NO: 2) (b) GTCCCGGAAGACGCTCAGGT;(SEQ ID NO: 3) (c) AAGGACGCAGCCACCATGCC; (SEQ ID NO: 4)(d) TTGGCGTAGCTCACCACGAA; (SEQ ID NO: 5) (e) AGATGCAGAAGACCACGAGG;(SEQ ID NO: 6) (f) CCACTCTGGAATGACACGGT; (SEQ ID NO: 8)(g) AGCATGGCCAGCAGGATGGA; (SEQ ID NO: 9) (h) GGCTGGAAGTTGAGTGCCAA;(SEQ ID NO: 11) (i) CTCAGGCTGTGGCTCTTTGG; (SEQ ID NO: 13)(j) AGCACGGCCCAGCCCCAGCC; (SEQ ID NO: 14) (k) GAGCTCCTTGAAGAAGACACT;(SEQ ID NO: 15) (l) CAGGATGGAGGAGATCCAGG; (SEQ ID NO: 17)(m) GAAGTTGAGTGCCAAACCCAA; (SEQ ID NO: 18) (n) CCCGTTGGCCATGGGGCGCC;(SEQ ID NO: 19) (o) GCCAGCCCGTTGGCCATGGG; (SEQ ID NO: 22)(p) CAGGGCACACAGGAAGACAG; (SEQ ID NO: 46) (q) ATGGCCAGGAGGGTTG;(SEQ ID NO: 47) (r) CATGGCCAGGAGGGTT; (SEQ ID NO: 48)(s) CCATGGCCAGGAGGGT; and (SEQ ID NO: 49) (t) CCCATGGCCAGGAGGG;

and wherein the ASO comprises a modified internucleoside linkage, amodified sugar moiety, or modified nucleobase, and wherein theoligonucleotide is not longer than 21 nucleotides in length.
 20. The ASOof claim 19, wherein the ASO comprises a modified internucleosidelinkage.
 21. The ASO of claim 20, wherein the modified internucleosidelinkage is a peptide-nucleic acid linkage, a morpholino linkage, a N3′to P5′ phosphoramidate linkage, a methylphosphonate linkage or aphosphorothioate linkage.
 22. The ASO of claim 19, wherein the ASOcomprises a modified sugar moiety.
 23. The ASO of claim 22, wherein themodified sugar moiety is 2′-O-alkyl oligoribonucleotide.
 24. The ASO ofclaim 19, wherein the ASO has a 2′MOE gapmer modification.
 25. The ASOof claim 19, wherein the ASO comprises a modified nucleobase.
 26. TheASO of claim 25, wherein the modified nucleobase is a 5-methylpyrimidine or a 5-propynyl pyrimidine.
 27. A pharmaceutical composition,the composition comprising (a) the antisense oligonucleotide (ASO) ofclaim 19; and (b) a pharmaceutically acceptable carrier.
 28. The ASO ofclaim 19, wherein the ASO has a sequence selected from the following:(SEQ ID NO: 1) (a) TCCCATGGCCAGGAGGGTTG; (SEQ ID NO: 5)(b) AGATGCAGAAGACCACGAGG; (SEQ ID NO: 46) (c) ATGGCCAGGAGGGTTG;(SEQ ID NO: 47) (d) CATGGCCAGGAGGGTT; (SEQ ID NO: 48)(e) CCATGGCCAGGAGGGT; and (SEQ ID NO: 49) (f) CCCATGGCCAGGAGGG.


29. The ASO of claim 28, wherein the ASO has a sequence selected fromthe following: (SEQ ID NO: 1) (a) TCCCATGGCCAGGAGGGTTG; (SEQ ID NO: 46)(b) ATGGCCAGGAGGGTTG; (SEQ ID NO: 47) (c) CATGGCCAGGAGGGTT;(SEQ ID NO: 48) (d) CCATGGCCAGGAGGGT; and (SEQ ID NO: 49)(e) CCCATGGCCAGGAGGG.


30. The ASO of claim 28, wherein the ASO has the sequence (SEQ ID NO: 5)AGATGCAGAAGACCACGAGG.


31. The ASO of claim 19, wherein the ASO has a sequence selected fromthe following: (SEQ ID NO: 3) (a) AAGGACGCAGCCACCATGCC; (SEQ ID NO: 4)(b) TTGGCGTAGCTCACCACGAA; (SEQ ID NO: 6) (c) CCACTCTGGAATGACACGGT;(SEQ ID NO: 9) (d) GGCTGGAAGTTGAGTGCCAA; (SEQ ID NO: 13)(e) AGCACGGCCCAGCCCCAGCC; and (SEQ ID NO: 14) (f) GAGCTCCTTGAAGAAGACACT.


32. The ASO of claim 31, wherein the ASO has the sequence (SEQ ID NO: 3)AAGGACGCAGCCACCATGCC.


33. The ASO of claim 31, wherein the ASO has the sequence (SEQ ID NO: 4)TTGGCGTAGCTCACCACGAA.


34. The ASO of claim 31, wherein the ASO has the sequence (SEQ ID NO: 6)CCACTCTGGAATGACACGGT.


35. The ASO of claim 31, wherein the ASO has the sequence (SEQ ID NO: 9)GGCTGGAAGTTGAGTGCCAA.


36. The ASO of claim 31, wherein the ASO has the sequence(SEQ ID NO: 13) AGCACGGCCCAGCCCCAGCC.


37. The ASO of claim 31, wherein the ASO has the sequence(SEQ ID NO: 14) GAGCTCCTTGAAGAAGACACT.


38. The ASO of claim 19, wherein the ASO has a sequence selected fromthe following: (SEQ ID NO: 2) (a) GTCCCGGAAGACGCTCAGGT; (SEQ ID NO: 8)(b) AGCATGGCCAGCAGGATGGA; (SEQ ID NO: 11) (c) CTCAGGCTGTGGCTCTTTGG;(SEQ ID NO: 15) (d) CAGGATGGAGGAGATCCAGG; (SEQ ID NO: 17)(e) GAAGTTGAGTGCCAAACCCAA; (SEQ ID NO: 18) (f) CCCGTTGGCCATGGGGCGCC;(SEQ ID NO: 19) (g) GCCAGCCCGTTGGCCATGGG; and (SEQ ID NO: 22)(h) CAGGGCACACAGGAAGACAG.


39. A method of treating prostate cancer in a subject, the methodcomprising admi nisteri ng to the subject a therapeutically effectiveamount of the antisense oligonucleotide (ASO) of claim
 1. 40. The methodof claim 39, wherein the ASO further comprises a modifiedinternucleoside linkage.
 41. The method of claim 40, wherein themodified internucleoside linkage is a peptide-nucleic acid linkage, amorpholino linkage, a N3′ to P5′ phosphoramidate linkage, amethylphosphonate linkage or a phosphorothioate linkage.
 42. The methodof claim 39, wherein the ASO further comprises a modified sugar moiety.43. The method of claim 42, wherein the modified sugar moiety is2′-O-alkyl oligoribonucleotide.
 44. The method of claim 39, wherein theASO has a 2′MOE gapmer modification.
 45. The method of claim 39, whereinthe ASO further comprises a modified nucleobase.
 46. The method of claim45, wherein the modified nucleobase is a 5-methyl pyrimidine or a5-propynyl pyrimidine.
 47. The method of claim 39, wherein the prostatecancer is characterized by elevated expression of MCT4.
 48. The methodof claim 10, wherein the prostate cancer is castration-resistantprostate cancer (CRPC).
 49. The method of claim 48, wherein the prostatecancer is enzalutamide (ENZ) resistant CRPC.
 50. The method of claim 39,wherein the ASO is administered intravenously.
 51. The method of claim39, wherein the ASO is topically administered to a tissue.
 52. Themethod of claim 39, wherein the ASO is mixed with lipid particles priorto administration.
 53. The method of claim 39, wherein the ASO isencapsulated in liposomes prior to administration.